Pharmaceutical formulations comprising vegetal material selected from trichilia

ABSTRACT

The present invention refers to a method for producing vasodilation or analgesia in a mammal, comprising the use of vegetal material derived from the bark of a Trichilia species.

FIELD OF THE INVENTION

The present invention refers to the use of vegetal material selectedfrom Trichilia for the preparation of pharmaceutical compositions.

BACKGROUND OF THE INVENTION

Medicinal plants known as catuaba have recognized pharmaceutical use dueto its aphrodisiac activities, as a tonic and in the treatment ofphysical and mental fatigues.

Phytotherapic formulations prepared from such plants extracts arealready known from the prior art, in which these specific extracts mayused alone or in association with other medicinal plant extracts, suchas the Brazilian shrub (Paullinia cupana). Several alternativeformulations comprising extracts from other catuaba species have alreadybeen disclosed in the art, all of them being related to the tonicactivity of this particular group of plants.

SUMMARY OF THE INVENTION

The present invention refers to the use of vegetal material selectedfrom Trichilia for the preparation of pharmaceutical formulationsshowing vasodilating and analgesic activities.

In another aspect the present inventions is directed to pharmaceuticalcompositions presenting vasodilating, analgesic or sexual stimulatingactivities comprising a vegetal material selected from Trichilia as theactive ingredient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effect of treatment with a Trichilia extract of malepatients suffering from functional sexual incapacity of varied etiology;

FIG. 2 shows the effect of L-NAME and L-Arg on basal tonus andrelaxation of the corpus carvernosum tissue of rabbits (RbCC);

FIG. 3 shows the effect of Atropine and TTX on RbCC basal tonus andrelaxation;

FIG. 4 shows the effect of Glybenclamide on RbCC basal tonus andrelaxation; and

FIG. 5 shows the effect of L-NAME on HCC basal tonus and relaxation.

DETAILED DESCRIPTION OF THE INVENTION

After detailed studies, the inventors found out that one particularplant of the group of catuaba, Trichilia sp., has surprising analgesicand vasodilating activities due to its vasorelaxant effect. It has beenfound that Trichilia catigua, for example, is able produce vasodilatingeffects in thoracic aorta and in pulmonary and mesenteric arteries ofrodents.

Although up to this moment there is no scientific corroboration of theexact mechanism which produces the above effect, evidences lead to theconclusion that the vasorelaxant activity of an Trichilia sp. extractdepends upon the presence or absence of endothetium in blood andlymphatic vessels. In this event, the above mentioned effect would beassociated to the release of nitric oxide (NO) or a NO-mediatedsubstance in the endothelium. Tests carried out in rats' aortas, forexample, showed that the vasorelaxation action of a Trichilia sp.extract was partially reduced when the vascular endothelium had beenremoved. In an additional experiment, the incubation of aorta, pulmonaryand mesenteric arteries of rats, rabbits and guinea-pigs with theselective NO-synthase inhibitor L-NOARG, N-nitro-L-arginine,significantly antagonized the vasorelaxation effect caused by Trichiliaextract. In a further assay, the inventors verified that the use ofmethylene blue which is an inhibitor of soluble guanylate cyclaseactivated by NO (Gruetter et al., 1981) partially affected thevasorelaxant action of Trichilia sp. in rings extracted from pulmonaryarteries.

Taken together, the results of the above cited studies show that thevasorelaxation actions of a vegetal material from Trichilia sp. ismediated both by endothelium-dependent and -independent mechanisms.

Due to the unexpected vasorelaxant activity of Trichilia sp., thepresent inventors found out that extracts thereof also show an analgesiceffect.

Tests carried out in chemical models of pain stimulation in mice, forexample, demonstrated a strong analgesic action of Trichilia extracts.Experimental results showed that this vegetal extract significantlyinhibits the nociceptive effect induced by the chemical agent—aceticacid 0.6% v/v. The material extracted from Trichilia produced asignificant and long lasting analgesic effect in the nociceptivecondition in accordance with the previously mentioned model and suchanalgesic effect may last up to 6 hours. On the other hand, additionaltests carried out with Trichilia extracts in association with otherplant extractive products demonstrated that when the animals arepreviously treated (10 minutes before the main administration) withnaloxone (a non-selective opioid antagonist), the analgesic action ofmorphine as well as of a Trichilia containing formulation has beenreverted in the acute and inflammatory phases of pain induced byformalin at 2.5% w/v as the chemical agent.

As occurs in its vasorelaxation action, the actual mechanism throughwhich Trichilia provides an analgesic effect is not totally explainedyet but it seems to involve, at least partially, an interaction with theopoid system. However, a deep analysis of the results obtained with theinvention indicates that vegetal extracts of this particular catuabaspecies present excellent effects in the treatment of pain alleviation.

An advantage of the present invention lays on the fact that, in contrastto what occurs, for example, with non-steroidal anti-inflammatory drugs,such as acetylsalicylic acid, Trichilia is effective in the initial(acute) phase of certain nociceptive conditions as it can be observed innocicepptive conditions induced by formalin and capsaicin.

Still another advantage of the present invention refers to the absenceof toxic effects caused by the vegetal material extracted from Trichiliasp. Tests carried out in mice, for example, clearly showed the lack oftoxicity in the administration of Trichilia sp. extracts, alone or inassociation with other plants extracts, even in high doses under acuteor subcronic forms, in daily doses up to 1 g/kg. This observation couldalso be verified in human beings in dosages of 25 mL twice a day forfour weeks.

Still in a further aspect, the present invention refers to the use of avegetal material selected from Trichilia sp. for the treatment offunctional sexual incapacity of varied etiology, liable to be clinicallytreated. A clinical test has been made in 14 male patients sufferingfrom functional sexual incapacity of varied etiology who were subjectedto a treatment with a liquid formulation comprising from 0.50 to 5.00%wt. of Trichilia sp. extract for 30 consecutive days demonstratedsignificant clinical recovery when evaluated for typical features of thesyndrome such as erection, orgasm, libido and ejaculation. The resultsare shown in FIG. 1. These results are supported by the pharmacologicalcharacterization of the dose-dependent vasorelaxant activity induced bythe formulation containing vegetal material selected from Trichilia inisolated segments of the corpus cavernosum both of rabbits and humanbeings. A complementary evaluation of probable mechanisms for thisparticular activity should encompass the verification of the possibilityof it to be involved with the ionic channels linked to the membrane ofthat tissue.

Therefore, the present invention is related to the vasoactive, analgesicand vasorelaxant activities of extracts of Trichilia and of formulationscontaining same as the active ingredient in connection with corpuscavernosum of vessels. In view of the proportionality of each of suchactivities in the final result due to the natural variability of thevegetal material and the dependence of the specific technologicalprocess applied for obtaining and transforming same, there may still bea functional inter-relationship among those activities. Thus, it isassumed that there is a contribution of the vasorelaxant effect in theanalgesic activity based on a better component distribution of theconstituents present in the vegetal material probably involved in thebiological mediation. On the other hand, there is a possiblerelationship of the analgesic action of Trichilia, particularly the oneprovided in the inflammatory phase of pain chemical induction, with thevasorelaxant effect mediated by the substances which take part in theinflammatory process, such as those analogous to cinines andprostaglandines.

The inventors have found out that any plant of the genus Trichilia, suchas T. catigua A. Juss, T. clausseni C. DC., T. casaretti C. DC., T.pallida Swartz. and T. elegans A. Juss, may be useful for the objectivesof the present invention. It has been verified that among the abovecited species, the Trichilia catigua is particularly preferred.Moreover, the vegetal material extracted from Trichilia sp. used in thepresent invention is preferably barks which are advantageously employedas alcoholic extracts and, more preferably, are formulated with inertpharmaceutically acceptable carries. Trichilia sp. formulations usefulfor the present invention may be, for example, orally administrated inthe form of tablets, coated tablets, soft and hard gelatin capsules,solutions, emulsions and suspensions, or rectally administrated in theform of suppositories. Suitable carriers include, but are not restrictedto, lactose, starch or its derivatives, talc, stearic acid or the saltsthereof in the case of solid formulations for oral administration.Carriers suitable for soft gelatin capsules, on the other hand, includevegetal oils, waxes, fats, liquid and semi-solid polyols. Solutions maybe prepared by using carriers selected from the group comprising water,carbohydrates and polyols. For suppositories appropriated carrierscomprise natural or hardened oils, waxes, fats and polyols.

In addition to the pharmaceutical carriers, the Trichilia spformulations in accordance with the present invention may also containadditives such as conservation agents, solubilizers, stabilizers,wetting agents, emulsifiers, sweeteners, dyers. flavoring agents,tonicity adjust aids, buffers, coating agents and antioxidants. They maystill comprise additional compatible active ingredients in the event itis desirable to enhance the vasodilating or analgesic action, or iffurther therapeutic effects are also intended.

In order to obtain the appropriate vasodilating or analgesic effects,the necessary dosage of Trichilia sp. will, of course, depend upon eachparticular case and may, therefore, vary in a wide range, being not adeterminant parameter of the present invention. However, it has beenobserved that an effective dosage for oral administration in humanbeings may vary in a range corresponding to 10 mg to 0.5 g of Trichiliasp. extract per day. In the event pharmaceutical formulations containingthe extract of Trichilia sp are used, the intended results may beeffectively obtained by employing concentrations of 0.2 to 50% wt. ofthe cited extract based on the total formulation.

The following illustrative examples will be useful better to demonstratethe present invention. It should be, however, noted that the followingillustrative data and procedures merely refer to some possibleembodiments of the invention which should not be taken as limitingparameters of the scope thereof.

EXAMPLES

Preparation of an Alcoholic Extract of Trichilia catigua

500 g of Trichilia catigua barks were extracted with an 1:1 (v/v)water-ethanol solution at room temperature for 7 days. The resultingextract was, then, filtered and concentrated using a rotavapor under 40mmHg at 60° C. The extract was maintained under refrigeration until use.

Preparation of Pharmaceutical Formulations

The Trichilia catigua alcoholic extract obtained as described above wasused to prepare the formulations set forth below:

Component % wt. Liquid Formulation (I) extract of Trichilia 0.50 a 5.00extracti of Muirapuama 0.10 a 2.00 extract of Gengibre 0.01 a 0.20extract of Guaraná 0.10 a 2.00 suitable carrier 90.80 a 99.29 SolidFormulation (II) extract of Trichilia  5 a 50 extract of Muirapuama  2 a15 extract of Gengibre 0.2 a 2   extract of Guaraná  2 a 15 suitablecarrier   18 a 90.80

Vasorrelaxant Activity Assay

(a) In Isolated Thoracic Aorta

Thoracic aortas isolated from male Wistar rats (250 g-350 g) werecleaned of connective tissue and adherent fat, without damaging theendothelium, cut into rings of about 2-3 mm wide and suspended in a 5 mLorganic bath chamber containing Krebs-Hanseleit solution at 37° C., pH7.2, gassed with 95% O₂ and 5% CO₂ at pH 7.4. For some experiments thevascular endothelium was removed by gently rubbing the internal surfaceof the artery with a wooden stick. Endothelium integrity wasfunctionally determined by evaluating the ability of acetylcholine (Ach1 μM) to produce relaxation of samples precontracted with noradrenaline(NA, 1-100 nM). The samples were considered as having a viableendothelium when Ach evoked relaxations of 60% and were considered asbeing endothelium denuded when Ach failed to cause relaxation (asdescribed in Furchgott and Zawadski, 1980).

Samples were submitted to a basal tension of 1 g and were allowed toequilibrate for at least 90 min prior to addition of any drug, the bathsolution being renewed every 20 min during this time period. Isometrictension changes were recorded by means of a F-60 force-transducer (NarcoBiosystems or Letica 6006). The submaximal contractile responses in thesamples induced by noradrenaline were taken as the 100% values and allsubsequent responses were calculated as a percentage of this value.

Following the equilibration period, samples were contracted by additionof noradrenaline (NA) (100 nM) and once the tonic responses becamestable (usually after 5 min), an alcoholic extract of Trichilia catiguaas previous prepared was cumulatively added both to the rings providedwith endothelium and to those from which the endothelium had beenremoved, at concentrations from 1 to 3000 μg/mL. Prior to itsutilization, the extract of Trichilia catigua was diluted until thealcohol concentration did not exceed 0.05% v/v in the nutrition medium.

The results of the cumulative addition of the extract of Trichiliacatigua did not show any effect in the tonus of the biologicalpreparations, but produced a vasodilating response both in the aortarings with intact endothelium and in the rings free from endothelium.The results are shown in Table 1:

TABLE 1 Effect of vegetal extract of Trichilia in aorta rings from ratsaorta rings of rats with endothelium without endothelium VegetalMaterial E_(max)(%) T. catigua 86.0 ± 7.1 28.0 ± 3.4 E_(max)(%) -Maximum Effect (%)

(b) In Isolated Pulmonary and Mesenteric Arteries

Isolated mesenteric and pulmonary arteries from guinea-pigs (400 g-450g) and rabbits (2 Kg-3 Kg) of both sexes were cleaned of connectivetissue and adherent fat, without damaging the endothelium, cut intorings of about 2-3 mm wide and treated in the same way describe in item(a) above.

Samples were submitted to a basal tension of 2-4 g, were allowed toequilibrate for at least 90 min prior to addition of any drug, the bathsolution being renewed every 20 min during this time period, and furthertreated as indicated in item (a) above.

The results of the cumulative addition of the extract of Trichiliacatigua did not show any effect in the tonus of the biologicalpreparations, but produced a vasodilating response in the pulmonary andmesenteric rings both with intact endothelium and without endothelium.

Analgesic Activity Assays

Male Swiss mice varying from 25 to 35 g, were housed at 22° C.±2° C.under a 12 h light/12 h dark circle and with access to water and purina.The animals were acclimatized to the laboratory for at least 1 hourbefore testing and were used once throughout the experiments.

Analgesia Assay

(a) Acetic Acid-induced Abdominal Constriction

The abdominal constriction induced by acetic acid injections (6.0% v/v)consists of the contraction of abdominal muscle together with thestretching of hind limbs. Animals were pretreated with 200 mg of analcoholic extract of Trichilia catigua 6 hours prior to the experiment.Control animals received a similar volume of a 0.90% NaCl solution (10mL/Kg). After challenge to the acetic acid, pairs of mice were placed inseparate boxes and the number of abdominal constrictions wascumulatively counted over a period of 20 minutes. Antinociception wasexpressed as the reduction of abdominal constrictions between controlanimals treated with the saline solution and mice pretreated with theTrichilia extract.

A composition comprising an extract of T. catigua associated with othervegetal extractive materials as set forth in formulation (I) previouslycited was tested in rats following the same procedure mentioned above.The results are indicated in Table 2:

TABLE 2 Analgesic Effect of Trichilla Vegetal Extractive Material inNociception Induced by Acetic Acid Nociception by Acetic Acid VegetalMaterial I_(max)(%) T. catigua 82.0 ± 2.0 Formulation with T. catigua(I) 70.0 ± 2.0 I_(max)(%) - Maximal Inhibition (%)

The data contained in the above table shows that the alcoholic extractof Trichilia catigua administrated alone or in a formulation in which itis associated with other vegetal extractive materials caused asignificant reduction of abdominal constrictions induced by acetic acid.

Formulation (I) comprising several vegetal extracts was additionallytested following the above procedure, but employing pretreatmentadministrations of 1 to 24 hours prior to the challenge to acetic acid.The results are shown in Table 3:

TABLE 3 Time-dependent Analgesic Effect of Extractive Formulation (I) ofTrichilia in the Nociception Induced in Mice by Acetic Acid TimeNociception by Acetic Acid (h) I_(max)(%) 1 29.6 ± 2.5 2 34.8 ± 1.5 457.8 ± 1.9 6 70.0 ± 2.0 8 43.2 ± 4.4 10   8.3 ± 2.1 12   3.8 ± 1.5 24  3.8 ± 2.9 I_(max)(%) - Maximal Inhibition (%)

(b) Formalin-induced Licking

Mice were lightly anaesthetized, with exception of those used to analyzethe first phase of fromalin-induced pain. The surface of the animalspaws was injected with 20 μL of 2.5% w/v formalin. Two groups of mice(mice treated with T. catigua and control mice) were simultaneouslyobserved over a period from 0 to 30 min after the formalin injection.The total time spent by the animal licking the injected paw was timedwith a chronometer and was considered as an indicative of pain. Thefirst phase of the nociceptive response normally occurred after 5 minafter the formalin injection and the second phase was from 15 to 30 minafter such induction. These phases represent the neurogenic andinflammatory responses, respectively. In order to avoid pharmacokineticinfluences, different groups of animals were used to analyze each phaseof the experiment. Alternatively, animals were pretreated with naloxone10 min prior to the administration of compositions comprising an extractof T. catigua. The obtained data is indicated in Table 4:

TABLE 4 Antinociceptive Effect of Vegetal Extract of Trichilia in PainInduced in Mice by Formalin nociception by formalin 0-5 min. 15-30 minVegetal Material I_(max)(%) T. catigua 32.0 ± 3.0 35.0 ± 10.0I_(max)(%) - Maximal Inhibition (%)

Superfusion of Isolated Corpus cavernosum

Corpus carvenosum tissue isolated from penis of New Zealand rabbits andadult men, cleaned of connective tissue and albuginic tunic wasdissected into segments of 2-3 cm wide. The samples were placed incascade form, under tension of 2.5 g, superfused in nutritiveKrebs-Ringer solution at 37° C. and pH of 7.2, gassed with 95% O₂ and 5%CO₂ at pH 7.4. The tissues were attached to auxotonic handles connectedto isotonic transducers (Harvard) for smooth muscles. The responses wereregistered in a multi-channel polygraph (Watanabe, WR 3101). Sampleswere stabilized for at least 90 min and the tissue tonus were induced bya continuous infusion (0.1 mL/min) of NA (3 μM) or KCI (35 mM). In thisexperiment, the agonists were administrated as a bolus (10-100 μL) whilethe antagonists and inhibitors were administrated by continuous infusion(0.1 mL/min) for about 20 min prior to the administration of formulation(I) comprising extractive material from Trichilia catigua. The resultsare indicated in FIGS. 2 to 5.

FIG. 2 shows that the infusion of L-NAME (N^(m)-nitro-L-arginine methylester, 10 μM) enhanced the basal tonus of the corpus cavernosum of therabbit (RbCC) and inhibited the relaxation induced by acetylcholine(ACh). Relaxations evoked by glyceryltrinitrato (GTN), and by thecomposition containing a vegetal material selected from Trichilia as theactive ingredient (Cat) were not affected by the L-NAME infusion. Thesubsequent infusion of L-arginine (L-arg, 300 μM) reverted the enhancedtonus and restored the relaxation induced by Ach. The experimental dataindicated in FIG. 2 is representative of 6 experiments.

FIG. 3, on the other hand, shows that the antagonist of muscarinicreceptors, atropine (1 μM) and sodium channel blockers, tetrodotoxin(TTX, 1 μM), did not present any effect on the relaxation of rabbits'corpus cavernosum (RbCC) induced by a composition containing a vegetalmaterial selected from Trichilia as the active ingredient (Cat) and byglyceryltrinitrate (GTN). However, relaxations evoked by theacetylcholine (Ach) and by poison of Tityus serrulaus (TSV) wereabolished by atropine and tetrodotoxin, respectively. The experimentaldata indicated in FIG. 3 is representative of 4 experiments.

In FIG. 4 it is possible to observe that the ATP-dependent potassiumchannel blocker (10 μM) inhibited the relaxation of the rabbit cavemousbody (RbCC) induced by cromakalim (CMK) without affecting the relaxationinduced by acetylcholine (Ach), by glyceryltrinitrate (GTN) and by thecomposition containing a vegetal material selected from Trichilia as theactive ingredient (Cat). The infusion of L-NAME enhanced the HCC tonusand significantly reduced the relaxation induced by Ach (0.6 μM). Therelaxations induced by GTN (1.3 μM) and the composition contain avegetal material selected from Trichilia as the active ingredient (Cat,10-100 μL) were not significantly affected by the L-NAME infusion. Forthese tests 4 tissue strips obtained from 2 donators were used.

What is claimed is:
 1. A method of producing vasodilation or analgesiain a mammal, comprising: administering to a mammal in need of suchtreatment an amount of a composition, derived from bark of a Trichiliaspecies selected from the group consisting of T. catigua A. Juss., T.clausseni C. DC., T. casaretti C. DC., T. pallida Swartz, and T. elegansA. Juss., sufficient to induce a vasorelaxant effect.
 2. The methodaccording to claim 1, wherein the Trichilia species is Trichiliacatigua.
 3. The method according to claim 1, wherein the compositionderived from a Trichilia species is a vegetal material in the form of analcoholic extract.
 4. The method according to claim 1, wherein theTrichilia species is a member selected from the group consisting of T.catigua, T. clausseni, T. casaretti, T. pallida, and T. elegans.